Multi-functional fluid flow device

ABSTRACT

The present invention relates to a fluid flow device. The device includes an elongate body having a proximal end, a distal end, and a length therebetween, at least one source fluid inflow port, at least one waste fluid outflow port, at least one well inlet port positioned at the distal end of the elongate body, at least one well outlet port positioned at the distal end of the elongate body, at least one conduit connecting the at least one source fluid inflow port to the at least one well inlet port, and at least one conduit connecting the at least one waste fluid outflow port to the at least one well outlet port.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. § 119(e)to U.S. Provisional Patent Application No. 62/311,654, filed Mar. 22,2016, the contents of which are incorporated by reference herein intheir entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numberAR063631 awarded by the National Institutes of Health (NIH). Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

Cells respond to their mechanical environment by activating biochemicalsignaling pathways, a process known as mechanotransduction.Mechanotransduction regulates diverse physiologic function in healthycells, and burgeoning evidence implicates dysregulation ofmechanotransduction cascades in disease pathology (Jaalouk, D. E.,Lammerding, J., 2009. Nat. Rev. Mol. Cell Biol. 10, 63-73). Given thatthese mechanotransduction pathways often integrate multiple signalingevents, there is a growing interest to identify key signaling nodes thatcan be targeted to modulate outputs that drive physiology or disease.

In the skeletal system, osteocytes sense mechanical strain within boneand respond with mechanotransduction signals (e.g., calcium (Ca2

), nitric oxide, extracellular ATP) and the expression of factors (e.g.,receptor activator of NFκB ligand (RANKL) and sclerostin) that controlthe activity of bone resorbing osteoclasts and bone forming osteoblasts(Bonewald, L. F., Johnson, M. L., 2008. Bone 42, 606-615; Klein-Nulend,J., Bakker, A. D., Bacabac, R. G., Vatsa, A., Weinbaum, S., 2013. Bone54, 182-190; Oftadeh, R., Perez-Viloria, M., Villa-Camacho, J. C.,Vaziri, A., Nazarian, A., 2015. J. Biomech. Eng. 137; Rubin, J., Rubin,C., Jacobs, C. R., 2006. Gene 367, 1-16; Thompson, W. R., Rubin, C. T.,Rubin, J., 2012. Gene 503, 179-193; Turner, C. H., Warden, S. J.,Bellido, T., Plotkin, L. I., Kumar, N., Jasiuk, I., Danzig, J., Robling,A. G., 2009. Sci. Signal. 2). Given that fluid flow through thelacunar-canalicular network generates shear stress drivingmechanotransduction in bone (Klein-Nulendetal, 2013), fluid flow is mostoften used to interrogate osteocyte response to mechanical load.

A number of commercially available systems have been developed to modelfluid flow in osteoblast and osteocyte-like cultured cells and each hasits own limitations, such as limited throughput and/or functionality (deCastro, L. F., Maycas, M., Bravo, B., Esbrit, P., Gortazar, A., 2015. J.Cell. Physiol. 230, 278-285; Espinha, L. C., Hoey, D. A., Fernandes, P.R., Rodrigues, H. C., Jacobs, C. R., 2014. Cytoskeleton 71, 435-445;Genetos, D. C., Geist, D. J., Liu, D., Donahue, H. J., Duncan, R. L.,2005. J. Bone Mineral. Res.: Off. J. Am. Soc. Bone Mineral. Res. 20,41-49; Michael Delaine-Smith, R., Javaheri, B., Helen Edwards, J.,Vazquez, M., Rumney, R. M., 2015. BoneKEy Rep. 4, 728) anddo-it-yourself (Aryaei, A., Jayasuriva, A. C., 2015. Mater. Sci. Eng. C,Mater. Biol. Appl. 52, 129-134; Burra, S., Nicolella, D. P., Francis, W.L., Freitas, C. J., Mueschke, N.J., Poole, K., Jiang, J. X., 2010. Proc.Natl. Acad. Sci. U.S.A. 107, 13648-13653; Shemesh, J., Jalilian, I.,Shi, A., HengYeoh, G., KnotheTate, M. L., Ebrahimi Warkiani, M., 2015.Lab. Chip 15, 4114-4127). Several of these systems have been developedto deliver physiologic fluid flow shear stress to osteoblast andosteocyte-like cells in culture. These systems range from simpleexperimental conditions where a droplet of buffer is released from apipette at a set height, to complex fluid flow systems such as theFlexCell Streamer.

While all the current models can deliver physiologically relevant fluidflow to cells in culture and generate physiologically relevant fluidshear stresses, each of these systems has their own disadvantages.Several of these systems require plating on proprietary slides, dishesor containers restricting the flexibility that the investigator has overplating surface conditions or geometries. Additionally, there is a largefinancial burden when purchasing these existing multi-component systemsrather than being able to integrate a fluid flow device into commonlyused cell culture systems and flow apparatus. Furthermore, many of thecurrently available flow systems are designed to only to test one sampleof cells at a time in a low-throughput manner rather than being able toconcurrently test multiple dishes or wells of cultured cells.

Thus, there is a need in the art for improved devices for culturingcells under physiological flow conditions offering high-throughputoptions with a cost-effective design. The present invention satisfiesthis unmet need.

SUMMARY OF THE INVENTION

A fluid flow device is described. The device includes an elongate bodyhaving a proximal end, a distal end, and a length therebetween, at leastone source fluid inflow port, at least one waste fluid outflow port, atleast one well inlet port positioned at the distal end of the elongatebody, at least one well outlet port positioned at the distal end of theelongate body, at least one conduit connecting the at least one sourcefluid inflow port to the at least one well inlet port, and at least oneconduit connecting the at least one waste fluid outflow port to the atleast one well outlet port.

In one embodiment, one of the at least one well inlet ports ispositioned in line with a central axis of the elongate body. In anotherembodiment, the at least one well inlet port extends from the distal endof the elongate body further than the at least one well outlet port. Inanother embodiment, the at least one well inlet port and the at leastone well outlet port extend the same distance from the distal end of theelongate body. In another embodiment, the at least one conduitconnecting the at least one source fluid inflow port to the at least onewell inlet port, and the at least one conduit connecting the at leastone waste fluid outflow port to the at least one well outlet port arepositioned within the elongated body. In another embodiment, the distalend of the elongate body is tapered. In another embodiment, the at leastone source fluid inflow port and the at least one waste fluid outflowport each extend outwardly from the length of the elongate body. Inanother embodiment, the at least one source fluid inflow port and the atleast one waste fluid outflow port each extend upward from the proximalend of the elongate body. In another embodiment, the device furthercomprises a flange positioned at a distal region of the elongate body,wherein the flange is capable of sealing the distal end of the elongatebody within a culturing well

Also described is a high-throughput fluid flow assembly. The assemblyincludes a plurality of fluid flow devices, wherein the fluid flowdevices are physically connected such that a central axis of eachelongate body of the fluid flow devices are parallel. In one embodiment,the source fluid inflow ports of each respective fluid flow device isfluidly connected to shared fluid source. In another embodiment, thewaste fluid outflow ports or each respective fluid flow device isfluidly connected to shared fluid reservoir.

Also described is a fluid flow kit. The kit includes at least one fluidflow device and an instruction material.

Also described is a method of applying fluid flow to biological cells ina culturing well. The method includes the steps of positioning a fluidflow device in a culturing well of a cell culture plate, such that theat least one well inlet port and at least one outlet port are positionedwithin the well, and flowing a fluid through the device of claim 1,wherein the fluid applies a shear stress to the cells in the culturingwell. In one embodiment, the step of flowing fluid through the fluidflow device further comprises a pump in line with the at least onesource fluid inflow port. In another embodiment, the flow rate of thefluid is adjustable. In another embodiment, the shear stress is about 4dynes/cm².

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of embodiments of the invention willbe better understood when read in conjunction with the appendeddrawings. It should be understood, however, that the invention is notlimited to the precise arrangements and instrumentalities of theembodiments shown in the drawings.

FIG. 1 is an isometric view of an exemplary fluid flow device (FFD) ofthe present invention.

FIGS. 2A and 2B are front and isometric views of a cell culturing platewith the FFD of FIG. 1 positioned within a well.

FIGS. 3A and 3B are front and bottom views of the FFD of FIG. 1 .

FIGS. 4A and 4B are front and bottom views of an alternative embodimentof an exemplary FFD of the present invention.

FIGS. 5A and 5B are front and bottom views of another alternativeembodiment of an exemplary FFD of the present invention.

FIG. 6A and FIG. 6B depict the results of experiments demonstrating themultiplexing capability of the FFD. FIG. 6A depicts an example of themultichannel peristaltic pump used when multiplexing the FFD and acomputer rendering of the FFD multiplexed in a single 96-well assayplate across 6 independent wells. FIG. 6B depicts western blot analysisof UMR106 cells simultaneously exposed to fluid flow in separate wells.Image J quantification of western blot analysis relative to GAPDH.Asterisks indicate statistical significance at p<0.05. Double asterisks(**) indicate statistical significance at p<0.01.

FIG. 7A through FIG. 7C depict the results of a computational fluiddynamics simulation. FIG. 7A illustrates simulated dynamic pressurewithin the FFD and well. FIG. 7B depicts simulated velocity within theFFD and well. FIG. 7C depicts simulated turbulence intensity within theFFD and well.

FIG. 8 illustrates simulated shear stress at the bottom surface of thewell. Computational trace of the average shear stress at the bottomsurface of the well over time. Data were determined using Solid Works2015 Flow Simulation software.

FIG. 9A through FIG. 9C depict vector lines indicating fluid flowtrajectories. FIGS. 9A and 9B illustrate trajectories of flow from thefront section view and a top view. FIG. 9C illustrates an up-close viewof the inlet and outlet trajectories within the well of the plate. Datawere determined using SolidWorks 2015 Flow Simulation software.

FIG. 10 through FIG. 12 depict the results of real-time Ca²⁺ imaging ofUMR106 cells during application of fluid flow. FIG. 10 depictsFluo-4Ca²⁺ indicator dye fluorescence before (basal) and during flow(response) of cells seeded in both the Ibidi chambers and a 96-wellplate with the FFD. Red line in image sequence indicates start andduration of fluid flow. Highlighted boxes indicate peak calciumresponses. FIG. 11 depicts traces of average Ca²⁺ indicator dyefluorescence intensity over time scaled to same basal level offluorescence intensity are shown in bold lines. Gray overlay tracesrepresent the Ca²⁺ response of individual cells in the well. FIG. 12depicts peak change in Ca²⁺ indicator dye fluorescence intensity andelevation/decay kinetics of Ca²⁺-dependent fluorescence. NS, notstatistically significant.

FIG. 13 illustrates western blot analysis of UMR106 cells exposed tofluid flow in the FFD. Fluid flow induced rapid activation of PKCsignaling and ERK signaling as indicated by an increase inphosphorylated PKC and phosphorylated ERK. Data are from the same geland exposure. Image J quantification of western blot relative to GAPDH.Double asterisks (**) indicate statistical significance at p<0.01.Triple asterisks (***) indicate statistical significance at p<0.001.

DETAILED DESCRIPTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value,as such variations are appropriate to perform the disclosed methods.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

DESCRIPTION

The present invention relates to a fluid flow device comprising inletand outlet ports, inflow ports and outflow ports, used for applyingshear stress to cultured cells. The invention provides a unique platformfor applying physiological shear stress and pressure to cultured cellsthat mimics the shear stress of the in vivo microenvironment. Theversatility in its function provides the fluid flow device with asignificant advantage over currently available models. The presentinvention can be used to image live cells in real-time during fluid flowby placing the device in an optics plate, such as an optically clearculture plate, or the like. Also, the present invention can form a fluidflow device assembly used for high-throughput molecular and biochemicalanalysis of cells following fluid flow. This is important whenconsidering potential variability between devices when performing avariety of experiments with differing output measures. By using the samedevice for every experiment rather than a device specifically forimaging and another device specifically for molecular and biochemicalanalyses, the possibility of obtaining variable results due to thedifferences in flow properties from one device to the next issignificantly reduced. Importantly, the device of the present inventionprovides a distinct improvement over current models by providing theability to multiplex the device. One can use multiple devices in thesame plate with a multichannel pump to obtain fully independentreplicates for each sample. Similarly, multiplexing allows for comparingmultiple samples that are subjected to identical flow under similarconditions at the same time. Lastly, a significant advantage of thepresent invention is the cost savings, in that the device and assemblydescribed herein offers a low-cost alternative to existing models.

Fluid Flow Device

Referring now to FIG. 1 , an exemplary fluid flow device (FFD) 100 ofthe present invention is shown. FFD 100 generally comprises an elongatebody 110 having a proximal end 101 and a distal end 102, a supportflange 112, a source fluid inflow port 114, a waste fluid outflow port117, a well inlet port 116, and a well outlet port 119.

Referring also to FIGS. 2A and 2B, elongate body 110 has a distal region111 that is sized to fit into a desired well 22 of a culturing plate 20,with the support flange 112 resting on top of the well plate surface toensure well inlet and outlet ports 116 and 119 are positioned fullywithin well 22 at the desired depth. In some embodiments, support flange112 may form a seal with the top opening of well 22. In someembodiments, distal region 111 may be tapered to provide a friction fit,or otherwise engage the opening of well 22 snuggly. In otherembodiments, the proximal region of elongate body 110 may be connectedto an automated or robotic system for placing and removing one or moreFFDs 100 into and out of the targeted wells 22 of culturing plate 20.

Distal region 111 of elongate body 110 may be any shape, provided itfits, loosely or snuggly, within the opening of the desired well 22. Theportion of elongate body 110 proximal to support flange 112 may be anydesired shape, such as cylindrical or rectangular, for example. Incertain embodiments, elongate body 110 proximal to support flange 112 issized and shaped so that multiple FFDs 100 can be efficiently stacked,such that each FFD 100 can be positioned in all or any number ofadjacent wells 22 within a multi-well culturing plate 20, for example asshown in FIG. 6 . Support flange 112 may likewise be any shape, providedthat it suitably extends beyond at least a portion of the perimeter ofthe desired well opening. In some embodiments, the overall length of FFD100 may be between 20 mm and 100 mm. In some embodiments, the overallouter diameter or width of FFD 100 may be between 10 mm and 200 mm.However, it should be appreciated that the overall size of FFD 100 isnot limited, and will be generally dictated according to the size of theculturing well or multi-well plate for which the FFD is designed for usewith.

As mentioned previously, FFD 100 further comprises a plurality of portsfor managing fluid flow from a fluid source, into the desired culturingwell, and out to a waste reservoir (or circulated through a common fluidreservoir). Referring now to FIGS. 3A and 3B, source fluid inflow port114 is fluidly connected to well inlet port 116 by an internal conduit115, thereby directing fluid to flow (block arrows) from a fluid sourceinto the culturing well. Likewise, well outlet port 119 is fluidlyconnected to waste fluid outflow port 117 by an internal conduit 118,thereby directing fluid to flow from the well to a waste reservoir orcommon fluid reservoir. In some embodiments, FFD 100 may include morethan one source fluid inflow port 114. In some embodiments, FFD 100 mayinclude more than one waste fluid outflow port 117. In some embodiments,FFD 100 may include more than one well inlet port 116. In someembodiments, FFD 100 may include more than one well outlet port 119. Insuch embodiments where more than one port of the same type is present,FFD 100 may likewise include more than one conduit 115 and/or 118, oralternatively conduits 115 and/or 118 may have multiple junctions tojoin or separate the flow from various ports. In some embodiments, oneor more valves (not shown) may be positioned in conduit 115 and/or 118,to regulate fluid flow by reducing or prohibiting fluid flowtherethrough.

Source fluid inflow port 114 and waste fluid outflow port 117 mayinclude barbs or ribbing on their exterior surface for engaging a softtubing or other conduit, such that each port is fluidly connected to afluid source and waste reservoir, respectively. Source fluid inflow port114 and waste fluid outflow port 117 may otherwise include a fittingthat is threaded, slip, compression, flare, flange, crimped, pressed,solvent welded, soldered, brazed, welded fitting, and the like. In oneembodiment, inflow port 114 and outflow port 117 may include fittingconfigurations comprising straight, elbow, coupling, union, reducer,cross, cap and plug, nipple, valve, tee connections, and the like.

It should be appreciated that inflow and outflow ports 114 and 117,respectively, may be positioned at any desired location on elongate body110 that is on, or proximal to, support flange 112. Further, inflow andoutflow ports 114 and 117, respectively, may be positioned at anydesired angle with respect to elongate body 110. For example, as shownin FIGS. 3A and 5A, inflow and outflow ports 114 and 117 may bepositioned such that they extend outwardly from a central region ofelongate body 110. In another embodiment, as shown in FIG. 4A, inflowand outflow ports 114 and 117, respectively, may be positioned such thatthey extend upward from the proximal end of elongate body 110.

Well inlet port 116 extends downward from the distal end of elongatebody 110. In some embodiments, inlet port 116 extends vertically intothe well, such that fluid flow exits inlet port 116 substantiallyperpendicular to a horizontal well floor. In other embodiments, inletport 116 extends at an angle between 1 degree and 90 degrees withrespect to the horizontal well floor. Similarly, well outlet port 119extends downward from the distal end of elongate body 110. In someembodiments, outlet port 119 extends vertically into the well, and inother embodiments, outlet port 119 extends at an angle between 1 degreeand 90 degrees with respect to the horizontal well floor. In someembodiments, well inlet port 116 extends into the culturing well furtherthan well outlet port 119, as is shown in FIG. 3A. In other embodiments,such as is shown in FIGS. 4A and 5A, inlet and outlet ports 116 and 119extend the same distance into the culturing well. In furtherembodiments, well outlet port 119 extends further into the culturingwell than well inlet port 116. Accordingly, in some embodiments, whenthe inlet port 116 extends further than the outlet port 119, FFD 100 maybe particularly suited for unidirectional flow through the furtherextending inlet port 116. Likewise, when inlet port 116 and outlet port119 extend the same distance, FFD 100 may be particularly suited formultidirectional flow. It should be appreciated that while the variousports are described here as inlets and outlets, there is no limitationto the final directional flow through the device. In all embodiments,both well inlet port 116 and well outlet port 119 have a length thatextends into the culturing well but do not make contact with thehorizontal well floor, such that the inlet and outlet ports 116 and 119do not touch any cellular or tissue growth on the horizontal well floorsurface. In some embodiments, the distance between the distal tip of thewell inlet port and the well floor is 2 mm to 4 mm. In some embodiments,the distance between the distal tip of the well outlet port 119 and thehorizontal well floor is 2 mm to 10 mm.

In some embodiments, well inlet port 116 may be centrally positioned,such that it is in line or concentric with a central axis 103 ofelongate body 110, as is shown in FIGS. 3A and 3B. As such, as fluidflows into the culturing well, it circulates radially from well inletport 116 and establishes laminar flow across the surface of theculturing well floor in which FFD 100 is placed, as is shown in FIGS. 9Aand 9B. In such embodiments, well outlet port 119 may be positionedadjacent to inlet port 116 and offset from the central axis. In otherembodiments, inlet and outlet ports 116 and 119 may be positionedadjacent to each other with a central axis 103 of elongate body 110running between ports 116 and 119, as is shown in FIGS. 4A, 4B, 5A and5B. In still other embodiments, well outlet port 119 may be centrallypositioned, such that it is in line with a central axis 103 of elongatebody 110, and well inlet port 116 may be positioned adjacent outlet port119 and offset from the central axis 103. It should be appreciated thatthere is no limitation to the positioning of well inlet and outlet ports116 and 119 relative to each other and relative to the central axis 103of elongate body 110.

It should also be appreciated that the openings to each port, as well asthe conduits between ports, can be of any desired shape and size,provided fluid can flow therethrough at the desired flow rate. Forexample, in some embodiments, the opening of source fluid inflow port114 may have an inner diameter of 1 mm to 2 mm. In some embodiments, theopening of waste fluid outflow port 117 may have an inner diameter of 1mm to 2 mm. In some embodiments, the opening of well inlet port 116 mayhave an inner diameter of 1 mm to 4 mm. In some embodiments, the openingof well outlet port 119 may have an inner diameter of 1 mm to 4 mm. Instill other embodiment one or more of ports 114, 116, 117 and 119 mayhave a tapered or expanded end or opening, thereby altering the innerdiameter and thus the flow rate of fluid therethrough. Likewise,conduits 115 and 118 may each have an inner diameter of 1 mm to 4 mm.Conduits 115 and/or 118 may have a constant inner diameter along itslength, or it may have a variable inner diameter along its length.

FFD 100 may be constructed from any suitable material, such as stainlesssteel, aluminum, polymers, plastics, and the like. Such materials mayoptionally be biocompatible or non-bioreactive materials, and may besuitable for any use with any type of culturing media or other desirableliquid. FFD 100 may be constructed as a single unit or a multi-componentassembled unit. FFD 100 or its component parts may be constructed viastandard molding, casting, printing or any other techniques understoodby those skilled in the art. In some embodiments, FFD 100 may beautoclavable, and in other embodiments it may be designed for single useand disposable.

Methods

The present invention also provides methods for shearing cultures ofcells using the various embodiments of the FFD as described herein. Inone embodiment, one or more FFDs is positioned in a well or wells of amulti-well plate having cells therein, such that the support flangesecures the FFD to the well, and optionally seals the well. In variousembodiments, the well is a well of a 384-well plate, a 96-well plate, a24-well plate, a 12-well plate, a 6-well plate, a single well plate, andthe like. A first tubing is connected to source fluid inflow port 114and a fluid source, and a second tubing is connected to waste fluidoutflow port 117 and waste reservoir. Alternatively, the first andsecond tubing can be connected to a common fluid reservoir to circulatethe same fluid through the FFD. In some embodiments, the tubing isfurther connected to and in line with a pump, for example a peristalticpump, a syringe pump, and the like. It should be appreciated that thechoice of pump used will be based on the experimental parametersrequired; thus, the system can be tuned to the specific flow parametersrequired for a specific cell type or experimental variable, as desired.In some embodiments, the fluid is driven by gravity. That is, a fluidsource reservoir connected to the inflow tubing is positioned at aheight that is greater than a fluid collection reservoir connected tothe outflow tubing. Accordingly, the difference in height of the tworeservoirs drives flow through conduits of the FFD, applying shearstress to cells cultured in the well.

In some embodiments, the method comprises applying a shear stress thatmimics a value for shear stress similar to what cells experience insitu. In some embodiments, a shear stress of 4 dynes/cm² is applied tothe cells using the FFDs described herein. In some embodiments, a shearstress of 20 dynes/cm² is applied to the cells. In some embodiments, ashear stress of 1 dynes/cm² to 4 dynes/cm², 4 dynes/cm² to dynes/cm², 10dynes/cm² to 15 dynes/cm², 15 dynes/cm² to 20 dynes/cm², 20 dynes/cm² to50 dynes/cm², or more than 50 dynes/cm² is applied to the cells. In someembodiments, the shear stress is adjusted by adjusting the fluid flowrate entering the FFD. In some embodiments, the shear stress is adjustedby adjusting the fluid flow rate at one or more points within a conduitand/or a port of the FFD. In some embodiments, the shear stress isadjusted by adjusting the viscosity of the fluid. It should beappreciated that the shear stress may additionally be adjusted by anyother means understood by those skilled in the art.

In some embodiments, the method includes applying physiological pressureto cultured cells. In some embodiments, the applied dynamic pressure isabout 1 dynes/cm². In some embodiments, the dynamic pressure is about750 dynes/cm². In some embodiments, the dynamic pressure is about 10dynes/cm², about 25 dynes/cm², about 50 dynes/cm², about 75 dynes/cm²,about 100 dynes/cm², about 250 dynes/cm², about 500 dynes/cm², and about750 dynes/cm². In some embodiments, the inlet dynamic pressure is about500 dynes/cm².

In some embodiments, for example when the FFD well inlet port iscentrally positioned concentric with the central axis of the FFD, andextends a greater distance into the culturing well than outlet port, auniform flow field is established directed radially from the well inletport, across the surface of the well floor, to the well outlet port,such that laminar flow is developed so that cells are sheared withlaminar or uniform shear stress. In some embodiments, for example whenthe FFD well inlet and outlet ports are adjacent, the central axisrunning between the inlet and outlet ports, and inlet and outlet portsextend the same distance into the culturing well, an oscillatory flowfield is established, and turbulent, nonlaminar or oscillatory flow isdeveloped so that cells are sheared with nonlaminar shear stress.

As shown in FIG. 6 , two or more FFDs can be used concurrently in amultiplexed array configuration. In some embodiments, the two or moreFFDs can use the same or separate fluid source reservoir and/or pumps.In some embodiments, the two or more FFDs are used for high-throughputmolecular and biochemical analysis of cells, such as ELISA, Westernblotting, mass spectrometry, and other assays understood by thoseskilled in the art. In some embodiments, the one or more FFDs are usedfor imaging studies, such as imaging with a microscope or other tool ortechnique known to one skilled in the art. In some embodiments, the oneor more FFDs are used for molecular and biochemical analysis of cellsand imaging studies simultaneously or in the same or similar experimentsthereby reducing experimental variability.

Kits

In one aspect, the present invention also includes a kit comprisinginstrumentation to apply fluid flow to cells plated in a culture plate.In certain embodiments, the kit is a sterile packaged kit. In certainembodiments, the one or more instruments of the fluid flow kit aresterile and contained in one or more individual sterile packages withinthe kit. The sterile fluid flow kit contemplated herein is thusimmediately ready for shear stress application upon removal of theinstruments from their respective packages without the need forpre-operation cleaning, sterilizing, or other processing. In certainaspects, the one or more instruments of the fluid flow kit aresingle-use instruments. For example, in one embodiment, the one or moreinstruments of the fluid flow kit are sterile and disposable. In anotherembodiment, the one or more instruments of the fluid flow kit arerepackaged after use, where, in certain embodiments, the one or moreinstruments may be reprocessed for future use. In one embodiment, theinstruments may be provided in one or more blister packaging. Eachblister may comprise a plastic container (e.g., PETG) component and alid (e.g., Tyvek®) component.

In certain embodiments, the fluid flow kit comprises at least one FFD asdescribed herein. In certain embodiments, the fluid flow kit mayoptionally include other instruments, such as and without limitation,one or more fittings, tubing, pumps, and/or reservoirs. For example, inone embodiment, the fluid flow kit comprises one or more fittings, whichmay be barbed fittings, threaded, slip, compression, flare, flange,crimped, and/or pressed fittings or the like, or a combination thereof.In one embodiment, the fluid kit comprises one or more lengths oftubing, including any size or geometry of tubing which may be necessaryfor the desired application. In one embodiment, the tubing is composedof Tygon®, nylon, polyethylene, or other suitable material known to oneskilled in the art. In one embodiment, the tubing comprises one or morestops for integration into holders of a peristaltic pump. In oneembodiment, the fluid flow kit comprises one or more pumps. In oneembodiment, the one or more pumps are peristaltic pumps, syringe pumps,or the like, as known to one skilled in the art. In one embodiment, thefluid flow kit may include one or more culturing wells or multi-wellplates. In one embodiment, the fluid flow kit may include culturingmedia or culturing media components for admixing. In one embodiment, thefluid flow kit may include a biological material, such as a cell ortissue.

In certain embodiments, the fluid flow kit is custom-configured withregard to size and geometry appropriate to the well being used. In oneembodiment, the kit is configured for a specific size of cylindricalbody or flange. In an alternative embodiment, the kit comprisesinstrumentation that may be necessary for various sizes of wells.

In certain embodiments, the kit comprises instructional material.Instructional material may include a publication, a recording, adiagram, or any other medium of expression which can be used tocommunicate the usefulness of any of the FFDs or fluid flow kitcomponents described herein. The instructional material may also includedescription of one or more steps to perform any of the methods describedherein. The instructional material of the kit may, for example, beaffixed to a package which contains one or more instruments which may benecessary for the desired procedure. Alternatively, the instructionalmaterial may be shipped separately from the package, or may be availableelectronically, such as accessible from the Internet, or anydownloadable electronic document file format.

Still further, the present invention includes a method of providing asterile fluid flow kit as described herein. In certain embodiments, themethod comprises receiving a request for one or more instruments for usein shearing cells or the like. In certain embodiments, the methodcomprises a customized request for particular instrumentation. Incertain embodiments, the method comprises a request for a standardizedkit which would contain the one or more instruments. In certaininstances, the method comprises gathering the one or more instrumentswhich were requested. In one embodiment, the method comprises processingthe one or more instruments. For example, in one embodiment, the one ormore instruments are sterilized. Sterilization of the one or moreinstruments may be conducted by any suitable method known in the art. Inone embodiment, the method comprises packaging the one or moreinstruments. For example, the one or more instruments may be packaged inone or more sterile packages to form a sterile kit.

It should be appreciated that the devices and the fluid flow kitsdescribed herein may be used for a variety of fluid flow or shear stressprocedures including but not limited to shearing cells, tissue,biological samples, non-biological samples, and the like. The proceduresmay be performed on any cell in the human or vertebrate body, including,but not limited to, osteocytes, osteoblasts, osteoclasts, endothelialcells, epithelial cells, smooth muscle cells, mesenchymal cells,progenitor cells, and the like. It should be understood that the presentdisclosure is not limited to a specific cell, tissue, or fluid flowapplication.

EXPERIMENTAL EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseExamples, but rather should be construed to encompass any and allvariations which become evident as a result of the teaching providedherein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the devices of the presentinvention and practice the claimed methods. The following workingexamples therefore and are not to be construed as limiting in any waythe remainder of the disclosure.

Example 1. Computational Fluid Dynamics Model

Computational Modeling

SolidWorks 2015 Flow Simulation software was used to perform computationfluid dynamics on the device and simulate the flow environment insidethe well during flow (FIGS. 7A-7C). The device was drawn to scale andcapped at the beginning of the inlet and the end of the outlet to form aclosed geometry. Two boundary conditions were set for the flowsimulation, inlet pressure and outlet flow volume. In order to determinethe pressure, the known inlet diameter and flow rate was used. First theflow velocity (cm/sec) was calculated using the known flow rate set bythe pump and area of the inlet (calculated from the known diameter)using Equation 1:

$\begin{matrix}{v = \frac{Q}{A}} & (1)\end{matrix}$Where v=velocity, Q=flow rate, and A=area of the inlet. Using thecalculated velocity the pressure was calculated in Pascals using theNavier-Stokes equation, simplified to the Bernoulli's equation, Equation2.

$\begin{matrix}{{\frac{1}{2}\rho V^{2}} + p + {\rho{gz}}} & (2)\end{matrix}$Where ρ=density, V=velocity, p=pressure, g=gravity, and z=height. Inorder for Bernoulli's principle to hold true for the system, thefollowing assumptions were made: (1) Flow buffer is a Newtonian fluid,(2) the flow is incompressible, and (3) there is no friction inside theflow.

For all simulations, the flow buffer was assumed to be similar to waterand thus, density=1000 kg/ml. The computational solver calculated flowvelocity (FIG. 8 ), flow trajectories (FIGS. 9A-9C), average shearstress at the bottom surface of the well, turbulence intensity (FIG.7C), and dynamic pressure (FIG. 7A) inside the well. It is important tonote, the boundary conditions used to run these simulations weredetermined from experimental data and what elicited the expectedbiological outcome based on published reports.

The results of the simulation indicate that the bottom surface of thewell where the cell monolayer resides is subjected to a nearly uniformaverage 4 dynes/cm² of shear stress (FIG. 8 ). Further, the velocitytrajectories indicate that the flow across the bottom surface of thewell is almost perfectly uniform such that the entire well of cells issubjected to the same forces. Interestingly, the turbulence intensitywithin the system is extremely low, 0.65%. These findings indicate thatthe flow system approximately mimics in vivo physiologic fluid shearstresses.

Device Validation

In order to validate the design of the fluid flow device, the biologicalresponse elicited by the FFD device was compared to other commerciallyavailable models. It is established that UMR106 osteoblast-like cellsare mechano-responsive and that fluid flow induces a rapid calciuminflux across the plasma membrane. Therefore, this cell line was used tocompare the device of the present invention to the commerciallyavailable and widely accepted Ibidi chamber slides for real-time livecell imaging. The Ibidi slides are known to produce laminar flow andbiologically relevant shear stress on cells cultured within thechambers. UMR106 cells were seeded in an Ibidi μ-slide I and loaded thecells with a calcium indicator dye, Fluo-4. Similarly, UMRI06 cells wereseeded into a 96-well plate with a special optics bottom. The sameGilson Minipuls 3 peristaltic pump was used for both conditions. Thepump flow rate was adjusted to achieve the same fluid shear stress aspreviously reported for the Ibidi chambers. When the response of UMRI06cells were compared in both conditions, an almost identical response wasshown (FIG. 10 ). Not only do both conditions show similar kinetics ofthe response (FIG. 11 ), but they also both show a similar peak changein fluorescence (FIG. 12 ).

To further validate the FFD design, the activation of fluid flow inducedsignaling was evaluated. It has been reported previously that fluid flowshear stress causes activation of ERK signaling in UMRI06 cells.Therefore, cells were seeded in a 96-well tissue culture treated plateand subjected the cells to 4 dynes/cm² of fluid shear stress. Afterflow, the cells were immediately lysed using a modified RIPA buffer.Whole cell lysates were then separated by SDS-PAGE for Western blotanalysis. It was observed that with the system of the present invention,fluid flow induces rapid activation of ERK signaling as indicated by anincrease is phosphorylated-ERK (FIG. 13 ).

These data suggest that the FFD design generates physiologic fluid flowshear stress conditions comparable to the accepted commerciallyavailable methods.

In conclusion, the fluid flow device of the present invention overcomesa variety of drawbacks that are associated with currently availablemodels. The FFD is highly versatile in its functionality and costssubstantially less. Also, the device delivers physiologically relevantfluid flow conditions to cells in monolayer culture. Furthermore, thedevice elicits similar biological responses in osteoblast-like cells asis commonly accepted in the field. Overall, the FFD provides an improvedmethod for studying the response of not only bone cells but anymechano-responsive cell type in vitro and will help advance the study ofmechanotransduction.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

What is claimed is:
 1. A method of applying fluid flow to biologicalcells in a culturing well, comprising: positioning a fluid flow devicein a culturing well of a multiwell cell culture plate, the devicecomprising an elongate tubular body having an upper proximal end, alower distal end, and a length therebetween, the distal end of theelongate tubular body configured for insertion into a well of amultiwell cell culture plate; at least one first source port positionedon the distal end; at least one first waste port positioned on thedistal end; at least one second source port positioned on the elongatetubular body above the at least one first source port; at least onesecond waste port positioned on the elongate tubular body above the atleast one first waste port; at least one source conduit positionedwithin the body connecting a single first source port out of the atleast one first source port(s) to a single second source port out of theat least one second source port(s); and at least one waste conduitpositioned within the body connecting a single first waste port out ofthe at least one first waste port(s) to a single second waste port outof the at least one second waste port(s), such that the at least onefirst source port and the at least one first waste port are positionedwithin the well; and flowing a fluid through the device; wherein thefluid applies a shear stress to the cells in the culturing well.
 2. Themethod of claim 1, wherein flowing fluid through the device furthercomprises activating a pump in line with the at least one second sourceport.
 3. The method of claim 2, wherein the flow rate of the fluid isadjustable.
 4. The method of claim 1, wherein shear stress is about 4dynes/cm².